Polynucleic acid-attached particles and their use in genomic analysis

Abstract

Disclosed are methods for preparing particle-linked polynucleotides, and using the particle linked polynucleotides in genomic analysis. The particles as disclosed are characterized as having a size variance of less than 2%.

1 . A method for preparing neutralized polynucleic acid-attached particles, comprising:
 size fractionating a plurality of charged surface-functionalized particles to provide a plurality of size-fractionated charged surface-functionalized particles being characterized as having a size variance of less than 2 percent;   neutralizing at least a portion of the size-fractionated charged surface-functionalized particles with a hydrophilic neutralizing agent to give rise to neutralized size fractionated particles being characterized as having a hydrophilic surface; and   attaching a polynucleic acid, directly or indirectly, to each of at least a portion of the neutralized size fractionated particles, or to each of at least a portion of the size-fractionated charged surface-functionalized particles, to provide neutralized polynucleic acid-attached particles.   
     
     
         2 . The method of  claim 1 , wherein each polynucleic acid is attached indirectly to at least a portion of the neutralized size-fractionated particles via a linker molecule attached to the size-fractionated charged surface-functionalized particles before neutralization. 
     
     
         3 . The method of  claim 1 , wherein each polynucleic acid is attached directly to at least a portion of the size-fractionated surface-functionalized particles via covalent bonding before neutralization. 
     
     
         4 . The method of  claim 1 , wherein the charged surface-functionalized particles are labeled or impregnated with one or more fluorescent dye molecules, energy-transfer donor-acceptor fluorescent dye molecule pairs, quantum dots, or any combination thereof. 
     
     
         5 . The method of  claim 4 , wherein the fluorescent dye molecules, energy transfer dye molecules, or quantum dots, or any combination thereof, emit at two or more different wavelengths with different intensities resulting in fluorescent pattern signatures. 
     
     
         6 . The method of  claim 1 , wherein the charged surface-functionalized particles are size fractionated using electrophoresis, chromatography, field-flow fractionation, or other general types of charge, size, or charge/size fractionation processes, or any combination thereof. 
     
     
         7 . The method of  claim 6 , wherein the electrophoresis uses an agarose gel, nanofabricated physical sieving device, or other sieving matrix, or any combination thereof. 
     
     
         8 . The method of  claim 1 , wherein the charged surface-functionalized particles comprise charged surface-functionalized latex particles, wherein the latex particles comprise a polymeric material composed of repeating units derived from styrenic monomers, acrylic monomers, acrylonitrile monomers, diene monomers, or any combination thereof. 
     
     
         9 . The method of  claim 1 , wherein the charged surface-functionalized particles comprise one or more functional groups grafted to the surface of the charged surface functionalized particles carboxylic acids, the functional groups comprising aliphatic amines, aromatic amines, hydrazide, sulfate, acidic functional groups, basic functional groups, or any combination thereof. 
     
     
         10 . The method of  claim 1 , wherein the plurality of charged surface-functionalized particles prior to fractionating are characterized as having an average particle size in the range of from about 5 nm to about 500,000 nm and a size variance distribution of greater than about 2%. 
     
     
         11 . The method of  claim 1 , wherein the plurality of size-fractionated, charged surface-functionalized particles are characterized as having an average particle size of greater than about 5 nm and a size variance of less than about 2 percent. 
     
     
         12 . The method of  claim 2 , wherein the linker molecules include difunctionalized primary amines for bonding to carboxy or other acidic surface modification, or primary amines comprising carboxylic acid for amine surface-modification, or any combination thereof,
 wherein the difunctionalized primary amines include any compound having the structure
   X′—Y—X″ 
   
       wherein X′ and X″ are functional groups and Y is an alkyl group comprising from 1 to about 40 carbon atoms and from 2 to about 80 hydrogen atoms. 
     
     
         13 . The method of  claim 12 , wherein X′ is a primary amine for attachment to the polynucleic acid and X″ is a carboxylate, amine, sulfate, or thiol group for attachment to the charged surface functional groups of the neutralized size-fractionated surface-functionalized particles. 
     
     
         14 . The method of  claim 1 , wherein the size-fractionated, charged surface-functionalized particles are neutralized by converting the charged surface functional groups to an uncharged hydrophilic group by reaction with a hydroxyl-bearing compound, amide-bearing compound, aldehyde-bearing compound, thiol-bearing compound, or any combination thereof. 
     
     
         15 . The method of  claim 14 , wherein the hydrophilic group includes a hydroxyl group, amide group, aldehyde group, thiol group, or any combination thereof. 
     
     
         16 . The method of  claim 15 , wherein the charged size-fractionated surface-functionalized particles are neutralized by reacting at least a portion of the charged surface functional groups with a hydroxyl-bearing compound, amide-bearing compound, aldehyde-bearing compound, thiol-bearing compound, or any combination thereof, and wherein the hydroxyl-bearing compound comprises 3-aminopropanol. 
     
     
         17 . The method of  claim 1 , wherein at least a portion of the size-fractionated charged surface-functionalized particles are neutralized, wherein particles that are less than fully neutralized are removed using electrophoresis. 
     
     
         18 . The method of  claim 1 , wherein the polynucleic acid comprises a PCR primer, a DNA fragment, a RNA fragment, or any combination thereof. 
     
     
         19 . A method, comprising:
 providing a plurality of primer-linked particles comprising PCR primer molecules, each labeled with a neutralized particle characterized as having an average particle size in the range of from about 5 nm to about 100 nm, each particle having a size variance of less than about 0.5 percent; and   amplifying a DNA sample using the primer-linked particles in the presence of PCR reagents to give rise to particle-labeled amplicons; and   performing size analysis of the particle-labeled amplicons using capillary electrophoresis.   
     
     
         20 . The method of  claim 19 , wherein the attached primers are one of the primers in a primer pair for amplification of STR loci used in forensic identification. 
     
     
         21 . The method of  claim 19 , wherein fewer than 10 thermocycles are performed to amplify the DNA. 
     
     
         22 . The method according to  claim 19 , wherein the capillary electrophoresis includes ELFSE comprising a device, the device comprising a capillary, a microfluidic channel, or any combination thereof, wherein the particle-labeled amplicons flow through a capillary or microfluidic channel length of between about 1 cm and about 100 cm, in the presence of an applied voltage field in the range of from about 50 V/cm to about 2000 V/cm. 
     
     
         23 . A polynucleic acid sample analysis reagent kit, comprising
 a plurality of size-fractionated, surface-neutralized particles having an average particle size in the range of from about 5 nm to about 500 microns, the size variance of the particles being less than about 0.5 percent, at least a portion of the particles being attached to a PCR primer or a linker molecule; and   a reagent to link a polynucleic acid to each of the linker molecules.   
     
     
         24 . The polynucleic acid analysis kit of  claim 23 , wherein the polynucleic acid comprises one or more DNA fragments obtained from a biological sample, or a synthetic PCR primer. 
     
     
         25 . A method of analyzing DNA fragments by size, comprising:
 attaching a plurality of surface neutralized particles to sample DNA fragments, wherein the surface neutralized particles have an average particle size in the range of from about 5 nm to about 500 microns, the size variance of the particles being less than about 0.5 percent, wherein at least a portion of said particles are attached to a PCR primer or linker molecule capable of being attached to a polynucleic acid using reagents to provide DNA-linked particles; and   analyzing the DNA-linked particles using ELFSE.